Compounds and methods for sequencing amino acids

ABSTRACT

The present invention provides methods and reagents for sequencing amino acids. One embodiment of the method for determining the terminal amino acid of a polypeptide comprises the steps of (a) attaching (either covalently or non-covalently) the polypeptide to a solid support, (b) reacting the polypeptide with a compound described below, under conditions and for a time sufficient for coupling to occur between the terminal amino acid of the polypeptide and the compound, thereby yielding a polypeptide with a derivatized terminal amino acid, (c) washing the solid support to remove unbound material, (d) cleaving the derivatized terminal amino acid from the polypeptide with a cleaving agent, (e) ionizing the cleaved derivatized terminal amino acid, and (f) determining the molecular weight of the derivatized terminal amino acid, such that the terminal amino acid is determined. 
     Within one embodiment, the compound is p-isothiocyanato phenethyl trimethylammonium and counterion salts thereof.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation-In-Part of U.S. Pat. No. 5,240,859,issued Aug. 31, 1993.

TECHNICAL FIELD

The present invention relates generally to proteins or polypeptides, andmore specifically, to compounds and methods which may be utilized todetermine the amino acid sequence of a protein or polypeptide.

BACKGROUND OF THE INVENTION

Proteins are among the most abundant of organic molecules, oftenencompassing as much as 50 percent or more of a living organisms dryweight. Proteins perform many different functions within a livingorganism. For example, structural proteins are often woven together inlong polymers of peptide chains to form fibrils, which are a majorconstituent of skin, tendon, ligaments, and cartilage. Proteins alsohave biological functions, including, for example, regulatory proteinssuch as insulin or growth hormones, protective proteins such asantibodies or complement, and transport proteins such as hemoglobin andmyoglobin. Many proteins are present only in very minute quantitieswithin living organisms, yet are nevertheless critical to the life ofthe organism. For example, loss of Factor VIII in humans leads tohemophilia, or the inability to properly clot blood.

Scientists have learned how to synthesize or express specific proteinsin order to therapeutically replace those proteins in individuals whoare deficient or lacking in the production of a particular protein. Inorder, however, to express these proteins from cells, or to artificiallysynthesize these proteins, it is first often necessary to determine theamino acid sequence of the protein.

Due in part to the great diversity of amino acids (there are at least 20different types found in naturally occurring proteins), it has been verydifficult to develop techniques suitable for sequencing proteins. Thisis partially due to the fact that some proteins may only be obtained invery small amounts. Thus, there has been a continuing need for improvedsensitivity in determining the sequence of amino acids in a protein.

Various methods have been suggested for the sequencing of proteins. Thefirst useful method for determining the amino-terminal (N-terminal) ofproteins was developed by Sanger, who found that the free, unprotonatedalpha-amino group of peptides reacts with 2,4-dinitrofluorobenzene(DNFB) to form yellow 2,4-dinitrophenyl derivatives (see Sanger andTuppy, Biochem. J. 49:463-490, 1961, see also Sanger and Thompson,Biochem. J. 53:353-374, 1963). Later methods were developed utilizing1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride), (seeGray and Hartley, Biochem. J. 89:379-380, 1963) resulting in a 100-foldincrease in sensitivity over Sanger's method. One difficulty with thismethod, however, is that it could only be performed once with the samesample of protein because the acid hydrolysis step destroys the protein,preventing analysis beyond the amino terminal amino acid of the protein.

In order to determine the identity of amino acids beyond the N-terminalamino acid residue, a widely used method for labeling N-terminal aminoacids (see Edman, Acta Chem. Scand 4:283, 1950) was applied tosequencing proteins. This method utilized phenylisothiocyanate to reactwith the free amino group of a protein, to yield the correspondingphenylthiocarbamoyl protein. Upon treatment with an anhydrous acid, theN-terminal amino acid is split off as an anilinothiazolinone amino acid,which is then convened to the corresponding phenylthiohydantoin (PTH)derivative. This PTH derivative may then be separated, and analyzed by,for example, liquid chromatography. Utilizing this method (Edmandegradation), repetitive cycles could be performed on a given peptideallowing the determination of as many as 70 residues in an automatedinstrument called a sequenator (see Edman and Begg, Eur. J. Biochem.1:80-91, 1967).

Currently, protein sequences are almost universally determined by Edmandegradation utilizing the reagent phenylisothiocyanate. The efficiencyand sensitivity of this process is, however, currently limited by theability of UV absorption to detect PTHs. Presently, the most sensitiveway to perform the Edman degradation is gas-liquid phase sequenceanalysis, where the polypeptides are non-covalently absorbed to asupport in a sequenator cartridge. This sequencing method allows theanalysis of protein and peptide sequences at the 10-20 picomole level.To reach that sensitivity level, the degradation chemistry must be tunedto an extent which does not allow for the recovery of PTH derivatives ofpost-translationally modified amino acids such as phosphate esters ofserine, threonine, or tyrosine residues. Even in cases where the site ofpost-translational modifications can be determined, with very fewexceptions, the nature of such modifications is generally notdeterminable. Current methods for determining the sites and nature ofpost-translational modification lag in sensitivity by approximately afactor of a thousand as compared to the capability of determiningpartial sequences. In addition, due to the complicated procedures forefficiently extracting contaminants and reaction by-products, thegas-liquid phase sequencing mode is prohibitively slow, requiring acycle time of 45 to 60 minutes.

There is, therefore, a need in the art for improved methods ofsequencing proteins or peptides which are present only in smallquantities. The present invention provides such a method, in partthrough the repetitive sequencing of extremely small quantities ofproteins or peptides (i.e., in the femtomole (10⁻¹⁵ moles) range), andfurther provides other related advantages.

SUMMARY OF THE INVENTION

The present invention provides compounds and methods suitable formicrosequencing very small quantities of polypeptides. Within one aspectof the present invention, a compound is provided, comprising, (a)anisothiocyanate group, (b) an ionizable group capable of detection bymass-spectrometry, and (c)a linker connecting the isothiocyanate groupwith the ionizable group. In one embodiment, the ionizable group is astrongly basic group discussed in more detail below. In anotherembodiment, the ionizable group is a strongly acidic group. In apreferred embodiment of the present invention, the compound isp-isothiocyanato phenethyl trimethylammonium and counterion saltsthereof. Representative counterion salts include halides such as iodide,bromide, chloride and fluoride, and oxyanions such as of acetate ortrifluoroacetate. In another preferred embodiment of the presentinvention, the compound is 4-(trimethyl aminopentylamidoethyl) phenylisothiocyanate and counterion salts thereof. In yet a further embodimentof the present invention, the compound is4-(3-pyridinylmethylaminocarboxypropyl) phenyl isothiocyanate.

Within another aspect of the present invention, a method for determiningthe terminal amino acid of a polypeptide is provided, comprising thesteps of (a) covalently or noncovalently attaching the polypeptide to asolid support, (b) reacting the polypeptide with a compound as discussedabove, under conditions and for a time sufficient for coupling to occurbetween the terminal amino acid of the polypeptide and the compound,thereby yielding a polypeptide with a derivatized terminal amino acid,(c) washing the solid support to remove unbound material, (d) cleavingthe derivatized terminal amino acid from the polypeptide with a cleavingagent, (e) ionizing the cleaved derivatized terminal amino acid, and (f)determining the molecular weight of the derivatized terminal amino acid,such that the identity of the terminal amino acid is determined. Withinone embodiment, subsequent to the step of determining the molecularweight of the derivatized terminal amino acid, steps (b) through (f) arerepeated such that the next amino acid is determined. The attachment ofthe polypeptide to the solid support may be either a covalent ornon-covalent attachment.

The present invention also provides a method for determining the aminoacid sequence of a polypeptide comprising the steps of (a) attaching thepolypeptide to a solid support; (b) reacting the polypeptide with acompound as described above, under conditions and for a time sufficientfor coupling to occur between the terminal amino acid of the polypeptideand the compound, thereby yielding a polypeptide with a derivatizedterminal amino acid; (c)washing the solid support to remove unboundmaterial; (d) cleaving the derivatized terminal amino acid from thepolypeptide with a cleaving agent; (e) ionizing the cleaved derivatizedterminal amino acid; (f) determining the molecular weight of thederivatized terminal amino acid; and (g) repeating steps (b) through (f)as recited above in order to determine the amino acid sequence of thepolypeptide. The attachment of the polypeptide to the solid support maybe either a covalent or non-covalent attachment.

Within a preferred embodiment, subsequent to the step of cleaving, thederivatized terminal amino acid is resolved such that derivatized aminoacids with identical molecular weights (such as leucine and isoleucine)are separated.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the mass spectrum for the phenethyl thiohydantoyltrimethyl ammonium trifluoroacetate derivative of isoleucine.

FIG. 2 illustrates the mass spectrum for the phenethyl thiohydantoyltrimethyl ammonium trifluoroacetate derivative of alanine.

FIG. 3 illustrates the mass spectrum for the phenethyl thiohydantoyltrimethyl ammonium trifluoroacetate derivative of glycine.

FIG. 4 illustrates the mass spectrum for the phenethyl thiohydantoyltrimethyl ammonium trifluoroacetate derivative of valine.

FIG. 5 illustrates the mass spectrum for 2.8 picomoles of the phenethylthiohydantoyl trimethyl ammonium trifluoroacetate derivative of valine.

FIG. 6 illustrates the mass spectrum for 2.8 picomoles of the phenethylthiohydantoyl trimethyl ammonium trifluoroacetate derivative of valine,for the mass range of 315 to 325 Da.

FIG. 7 illustrates the mass spectrum for 280 femtomoles of the phenethylthiohydantoyl trimethyl ammonium trifluoroacetate derivative of valine,for the mass range of 315 to 325 Da.

FIG. 8 illustrates the mass spectrum for 28 femtomoles of the phenethylthiohydantoyl trimethyl ammonium trifluoroacetate derivative of valine,for the mass range of 315 to 325 Da.

FIG. 9 is a table listing the predicted molecular weight for commonamino acid PETMA phenylthiohydantoin (PETMA-PTH) derivatives.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides compounds and methods forsequencing very small quantities of protein or polypeptide. Within thecontext of the present invention, the term "polypeptide" is understoodto include proteins as well as peptide chains of 2 or more amino acids.Generally, a compound of the present invention comprises (a) anisothiocyanate group, (b) an ionizable group capable of detection bymass spectrometry, and (c) a linker connecting the isothiocyanate groupwith the ionizable group.

Isothiocyanate groups (N═C═S) of the present invention are well known inthe art (see Doolittle, "An Anecdotal Account of the History of PeptideStepwise Degradation Procedures," Methods in Protein Sequence Analysis,Elzinga (ed.), Humana Press, Clifton, N.J.). The isothicyanate group isthe functional group of the compound, and is reacted with the N-terminalamino acid from a polypeptide under basic conditions, such that athiocarbamoyl derivative is formed (see Edman, Acta Chem. Scand 4:283,1950). As discussed in more detail below, this derivative is thencleaved from the polypeptide, preferably with an acidic cleaving agent.

The isothiocyanate group is separated from the ionizable group by alinker. The linker is designed such that it is chemically stable andinert, and such that it allows efficient separation of theisothiocyanate group and the ionizable group (i.e., allows theisothiocyanate group and ionizable group to react independently). Withina preferred embodiment of the invention, the linker is composed of ahydrocarbon chain or, most preferably, of a hydrocarbon chain linked toa phenyl ring. Within the latter embodiment, the phenyl ring, which ispositioned next to the isothiocyanate group, has a desirable electronicstructure which allows for optimal coupling and cyclization/cleavagerates. The hydrocarbon chain which is positioned next to the ionizablegroup provides additional separation between the ionizable group and theisothiocyanate group. As will be understood by one of ordinary skill inthe art, a virtually limitless array of hydrocarbon chains and modifiedhydrocarbon chains may be utilized within the present invention.Preferred hydrocarbon chains which are attached to the phenyl ring maybe found in the family of alkanes, with particularly preferred linkersranging from 2 carbon atoms to about 20 carbon atoms in length. Within apreferred embodiment of the invention, the linker is a phenethyl group.

Within another preferred embodiment of the present invention, the linkeris more hydrophobic than a phenethyl linking group. Such hydrophobiclinkers impart hydrophobic character to the polypeptide sample to besequenced and subsequently to the derivatized terminal amino acid to beextracted and identified. In particular, the hydrophobic nature impartedto the polypeptide renders compounds bearing these hydrophobic linkersparticularly well suited to adsorptive sequence analysis, i.e., sequenceanalysis of polypeptides which are non-covalently attached to a solidsequencing support. In a preferred embodiment, the hydrophobic linker isa phenethylamidopentyl group. The effect of the amidopentyl group is toincrease the hydrophobicity relative to the phenethyl group. Compoundsof the present invention which bear hydrophobic linkers such as thosedescribed above also serve to increase the efficiency of extraction ofthe derivatized terminal amino acid by the organic extraction solventfrom the remaining polypeptide attached to the solid sequencing support.The hydrophobic linkers increase the solubility of the derivatizedterminal amino acids in the extraction solvent, and thereby increase theefficiency of extraction which may increase the yield of product that isdetected in the sequencing method.

As noted above, the ionizable group is selected such that it is capableof detection by mass spectrometry. Within the context of the presentinvention, groups which are 0.1% to 1% ionizable are preferred; groupswhich are 1% to 10% ionizable are particularly preferred; and groupswhich are greater than 10% ionizable are most particularly preferred. Aparticularly preferred compound, p-isothiocyanato phenethyl trimethylammonium chloride (PETMA-PITC) is 10% to 50% ionizable. Anotherparticularly preferred compound is 4-(trimethylaminopentylamidoethyl)phenyl isothiocyanate (C5 PETMA-PITC). Such ionization efficiencies maybe readily calculated by one of ordinary skill in the art utilizingstandard techniques (see Smith et al., Anal. Chem. 60:1948, 1988).

Many different compounds are ionizable, and thus suitable for detectionby mass spectrometry, including, for example, the salts of strong acidsor strong bases. Within the context of the present invention, a "strongacid" includes those acids with a pKa of less than 4, and preferablyless than 2, and most preferably less than 1. A "strong base" includesthose with a pKa of greater than 8, preferably greater than 10, and mostpreferably greater than 12. Representative examples of salts of strongacids include phosphate salts such as sodium phosphate and potassiumphosphate, sulfate salts such as sodium sulfate, potassium sulfate,ammonium sulfate, or sulfonates such as potassium sulfonate.Representative examples of salts of strong bases include ammonium saltssuch as ammonium chloride, and quaternary amines such astrimethylammonium chloride. As will be understood by one of ordinaryskill in the art, the strong acids or bases discussed above areaccompanied by various counterion salts. For example, within variousembodiments the counterion salt for an acid may be sodium or potassium.In like manner, many counterion salts for strong bases, such astrimethylammonium are known. Representative examples include halidessuch as fluoride, chloride, bromide, or iodide, or oxyanions such as ofacetate or trifluoroacetate.

Alternatively, the ionizable group may be a neutral, uncharged group or,a weak base in solution, either of which acts as a strong base in thegas phase. Suitable groups include compounds which bear a lone pair ofelectrons, such as amines or heterocyclic aromatic compounds. In oneembodiment, the ionizable group is a pyridyl group. In a particularlypreferred embodiment, the ionizable group is4-(3-pyridinylmethylaminocarboxypropyl) phenyl isothiocyanate(PITC-311).

Once the isothiocyanate group, linker, and ionizable groups have beenselected, the final compound may be synthesized by one of ordinary skillin the art utilizing standard organic chemistry reactions. As notedabove, a preferred compound for use within the present invention isPETMA-PITC. This compound retains the excellent characteristics ofphenylisothiocyanate in the coupling and cyclization/cleavage reactionsof Edman degradation. Furthermore, the compound performs well inEdman-type chemistry because the electron structure of the phenyl ringis sufficiently separated from the quaternary ammonium group by theethyl linker, thus allowing the isothiocyanate to react undisturbed bythe quaternary ammonium group. Preparation of PETMA-PITC is describedbelow in Example 1. The preparations of C5 PETMA-PITC and PITC-311 aredescribed below in Examples 5 and 7, respectively.

The coupling and cyclization/cleavage rates of the compound may betested to ensure that the compound is suitable for sequencingpolypeptides. As an example, measurement of coupling andcyclization/cleavage rates for the compound PETMA-PITC is set forthbelow in Example 2. In general, the faster the coupling rate the morepreferred the compound. Coupling rates of between 2 and 10 minutes at50° C. to 70° C. are particularly preferred. Compounds which take longerthan 15 minutes for complete coupling are less desirable due to thelength of time it would take to run several sequential amino aciddegradation reactions. Similarly, fast cyclization/cleavage rates arealso preferred, because exposure to an acid over an extended period oftime will hydrolyze the peptide bonds in the polypeptide. Preferably,the cyclization/cleavage reaction should be essentially complete in 5minutes or less after incubation at 50° C.

Once a suitable compound has been selected, the compound may be utilizedto determine the terminal amino acid of a polypeptide. Briefly, thismethod comprises the steps of: (a)covalently or non-covalently attachingthe polypeptide to a solid support; (b) reacting the polypeptide with acompound as discussed above, under conditions and for a time sufficientfor coupling to occur between the terminal amino acid of the polypeptideand the compound, thereby yielding a polypeptide with a derivatizedterminal amino acid, (c) washing the solid support to remove unboundmaterial, (d) cleaving the derivatized terminal amino acid from thepolypeptide with a cleaving agent, (e) ionizing the cleaved derivatizedterminal amino acid, and (f) determining the molecular weight of thederivatized terminal amino acid, such that said terminal amino acid isdetermined. Additionally, steps (b) through (f) may be repeated suchthat the entire amino acid sequence of the polypeptide is determined.The determination of the N-terminal amino acid of a syntheticdecapeptide using PITC-311 is described below in Example 8.

As noted above, methods of the present invention may be utilized inorder to determine the sequence of a polypeptide. Within preferredembodiments of the invention, the polypeptide is "substantially pure,"which means that the polypeptide is about 80% homogeneous, andpreferably about 99% or greater homogeneous. Many methods well known tothose of ordinary skill in the art may be utilized to purify thepolypeptide prior to determining its amino acid sequence. Representativeexamples include HPLC, Reverse Phase--High Pressure LiquidChromatography (RP-HPLC), gel electrophoresis, chromatography, or any ofa number of peptide purification methods (see generally the series ofvolumes entitled "Methods in Protein Sequence Analysis"). Gelelectrophoresis (see Aebersold, J. Biol. Chem. 261(9):4229-4238, andRP-HPLC (see Aebersold, Anal. Biochem. 187:56-65, 1990) are particularlypreferred purification methods. Additionally, for sequencing purposes,polypeptides of from 3 to 50 amino acids in length are preferred, withpolypeptides from 10 to 30 amino acids being particularly preferred.Proteins or polypeptides may readily be cleaved into preferred length bymany methods, including, for example, by chemical methods, by enzymaticmethods, or by a combination of the two. Representative chemicalcompounds which may be utilized to cleave proteins or polypeptidesinclude cyanogen bromide, BNPS-skatole, and hydroxylamine (all availablefrom Aldrich Chemical Company, Milwaukee, Wis.). Representative enzymesinclude trypsin, chymotrypsin, V8 protease, or Asp N (all available fromBoehringer Mannheim Biochemicals, Indianapolis, Ind.). The mixture ofcleaved fragments may then be separated to like sized fragments byvarious methods, including, for example: gel electrophoresis, HPLC, andRP-HPLC. Reversed-phase HPLC is particularly preferred for theseparation of these fragments due to its capability of high resolution.

Although substantially pure polypeptides are preferably utilized withinthe methods described herein, it is also possible to determine thesequence of polypeptide mixtures. Briefly, an algorithm may be utilizedin order to determine all of the hypothetical sequences with acalculated mass equal to the observed mass of one of the peptides in themixture. (See Johnson and Walsh, Protein Science 1:1083-1091, 1992).These sequences are then assigned figures of merit according to how welleach of them accounts for the fragment ions in the tandem mass spectrumof the peptide (see Johnson and Walsh, supra) utilizing such algorithms,the sequence of polypeptides within the mixture may be readilydetermined.

The polypeptide is then attached (either covalently or non-covalently)to a solid support for protein sequencing. Various materials may be usedas solid supports, including, for example, numerous resins, membranes orpapers. These supports may additionally be derivatized to facilitatecoupling. Supports which are particularly preferred include membranessuch as Sequelon™ (Milligen/Biosearch, Burlington, Mass.).Representative materials for the construction of these supports include,among others, polystyrene, porous glass, polyvinylidinefluoride andpolyacrylamide. In particular, polystyrene supports include, amongothers: (1)a (2-aminoethyl) aminomethyl polystyrene (see Laursen, J. Am.Chem. Soc. 88:5344, 1966); (2) a polystyrene similar to number (1) withan aryl amino group (see Laursen, Eur. J. Biochem. 20:89, 1971);(3)amino polystyrene (see Laursen et al., FEBS Lett. 21:67, 1972); and(4)triethylenetetramine polystyrene (see Horn and Laursen, FEBS Lett.36:285, 1973). Porous glass supports include: (1) 3-aminopropyl glass(see Wachter et al., FEBS Lett. 35:97, 1973); and(2)N-(2-aminoethyl)-3-aminopropyl glass (see Bridgen, FEBS Left. 50:159,1975). Reaction of these derivatized porous glass supports withp-phenylene diisothiocyanate leads to activated isothiocyanato glasses(see Wachter et al., supra). Polyacrylamide-based supports have alsobeen utilized, including a cross-linked β-alanylhexamethylenediaminepolydimethylacrylamide (see Atherton et al., FEBS Lett. 64: 173, 1976),and an N-aminoethyl polyacrylamide (see Cavadore et al., FEBS Lett. 66:155, 1976).

One of ordinary skill in the art may then readily utilize appropriatechemistry to couple the polypeptide to the solid supports describedabove (see generally Machleidt and Wachter, Methods in Enzymology: [29]New Supports in Solid-Phase Sequencing 263-277, 1974). Preferredsupports and coupling methods include the use of aminophenyl glass fiberpaper with EDC coupling (see Aebersold et al., Anal. Bioch. 187:56-65,1990); DITC glass filters (see Aebersold et al., Biochem. 27:6860-6867,1988) and the membrane polyvinylidinefluoride (PVDF) (Immobilon P ™,Milligen/Biosearch, Burlington, Mass.), along with SequeNet™ chemistry(see Pappin et al., Current Research in Protein Chemistry, VillafrancaJ. (ed.), pp. 191-202, Academic Press, San Diego, 1990).

In the practice of the present invention, attachment of the polypeptideto the solid support may occur by either covalent or non-covalentinteraction between the polypeptide and solid support. Thus, in additionto the solid-phase (covalent) sequencing techniques discussed above,sequencing techniques with non-covalent sample immobilization, such as,for example, liquid-phase, gas-liquid-phase, gas-phase,pulsed-liquid-phase and absorptive sequencing techniques, may also beemployed. For non-covalent attachment of the polypeptide to the solidsupport, the solid support is chosen such that the polypeptide attachesto the solid support by non-covalent interactions. For example, a glassfiber solid support may be coated with polybrene to provide a solidsupport surface which will non-covalently attach the polypeptide. Othersuitable adsorptive solid phases are commercially available. Forexample, polypeptides in solution may be immobilized on syntheticpolymers such as polyvinylidine difluoride (PVDF, Immobilon, MilliporeCorp., Bedford, Mass.) or PVDF coated with a cationic surface (ImmobilonCD, Millipore Corp., Bedford, Mass.). These supports may be used with orwithout polybrene. Alternatively, polypeptide samples can be preparedfor sequencing by extraction of the polypeptide directly frompolyacrylamide by a process called electroblotting. The electroblottingprocess eliminates the isolation of polypeptide from other peptideswhich may be present in solution. Suitable electroblotting membranesinclude Immobilon and Immobilon CD (Millipore Corp., Bedford, Mass.).

When the polypeptide is attached to the solid support throughnon-covalent interactions, the physical properties of the compound maybe selected to optimize sequencing. For example, the compound PETMA-PTHfunctions best when the polypeptide is covalently attached to the solidsupport. Since PETMA-PTH is a very polar compound, the subsequentwashing step is preferably accomplished with a very polar washingsolvent. While very polar washing solvents do not substantially removecovalently bound polypeptide from the solid support, such solvents mayremove non-covalently attached polypeptide. When the polypeptide isattached to the solid support by non-covalent interactions, it isdesirable to use a less polar washing solvent, and thus a less polarcompound. Such compounds can readily be synthesized by one skilled inthe art by standard organic synthesis techniques. For example, a skilledartisan could modify either the linker group, ionizable group, or both,to yield a compound having the desired polarity. Both C5 PETMA-PITC andPITC-311, by virtue of their hydrophobic nature (i.e., less polar thanphenethyl linking groups), are well-suited for adsorptive polypeptidesequencing, i.e., sequencing involving the non-covalent attachment ofthe polypeptide to the sequencing support. In both compounds, the linkergroups are more hydrophobic than in PETMA-PITC, and in PITC-311, theionizable group, pyridyl, is also hydrophobic. The non-polar nature ofC5 PETMA-PITC and PITC-311 permits the use of non-polar washing andextraction solvents which in turn allows the use of adsorptivepolypeptide sequencing without the removal and loss of the polypeptideto be sequenced from the solid sequencing support.

The polypeptide which is attached to the solid support may now bereacted with a compound as described above, under conditions and for atime sufficient for coupling to occur between the terminal amino acid ofthe polypeptide and the compound, thereby yielding a polypeptide with aderivatized terminal amino acid. As discussed above, it is preferred toconduct preliminary experiments with the compound to determine thepreferred time and conditions in order to best effect coupling. In thecase of PETMA-PITC, as demonstrated in Example 2A, 66% coupling wasachieved with this compound at a concentration of 0.8%, after 20 minutesat 50° C. In a preferred embodiment of the present invention, theconcentration of PETMA-PITC is increased to greater than 1%.

Preferably, coupling occurs under basic conditions, for example, in thepresence of an organic base such as trimethyl amine, triethyl amine, orN-ethylmorpholine. In a preferred embodiment, the compound PETMA-PITC isallowed to couple with the bound polypeptide in the presence of 5%N-ethylmorpholine in methanol: H₂ O(75:25 v/v).

Subsequently, the solid support is washed to remove all unboundmaterial, including uncoupled compound, excess coupling base, andreaction byproducts. Various reagents are suitable as washing solvents,including, for example, methanol, water, a mixture of methanol andwater, or acetone. For less polar reagents, less polar washing solventsare preferred, such as acetonitrite, heptane, ethylacetate andchloroform.

The derivatized terminal amino acid which is now bound to the compoundmay then be cleaved from the rest of the bound polypeptide, preferablywith a strong acid which is utilized as the cleaving agent. Variousstrong acids are suitable for use within the present invention,including, for example, trifluoroacetic acid, heptafluorobutyric acidand hydrochloric acid. Within a preferred embodiment, 100%trifluoroacetic acid is utilized to cleave the derivatized terminalamino acid from the polypeptide.

Within the present invention, the cleaved derivatized terminal aminoacid is then ionized, and the molecular weight determined by a massspectrometer. Various mass spectrometers may be used within the presentinvention. Representative examples include: triple quadrupole massspectrometers, magnetic sector instruments (magnetic tandem massspectrometer, JEOL, Peabody, Mass.), ion-spray mass spectrometers,Bruins et al., Anal. Chem. 59:2642-2647, 1987 electrospray massspectrometers, Fenn et al., Science 246:64-71, 1989, laser desorptiontime-of-flight mass spectrometers, Karas and Hillenkamp, Anal. Chem.60:2299-2301, 1988, and a Fourier Transform Ion Cyclotron Resonance MassSpectrometer (Extrel Corp., Pittsburgh, Mass.). Within a preferredembodiment, a triple quadrupole mass-spectrometer with an electron-sprayor ion-spray ionization source (model API III, SCIEX, Thornhil, Ontario,Canada) is utilized to ionize the derivatized terminal amino acid, andto determine its molecular weight. If the terminal amino acid isderivatized with the preferred compound PETMA-PITC, the ionizable grouptrimethylammonium chloride mediates excellent ionization in electronspray ionization sources. This compound allows detection of femtomolequantities of PETMA-PTH derivatives. Similar results are obtained whenC5 PETMA-PITC and PITC-311 are used.

Within all embodiments described herein, the steps of reacting thepolypeptide through determining the molecular weight of the derivatizedterminal amino acid may be repeated such that the next amino acid in thepolypeptide is determined.

Within one aspect of the present invention, the above methods areautomated. Instruments such as the Milligen/Biosearch 6600 proteinsequenator may be utilized along with the reagents discussed above, inorder to automatically sequence the polypeptide. This instrument has thenecessary programming flexibility to develop new degradation cyclesoptimized for the novel compound, and is particularly preferred forcovalent sequencing. For non-covalent attachment sequencingapplications, a variety of other sequenators are preferably utilized,including, for example, Models 477 or 473 (ABI, Foster City, Calif.) andBeckman/Porton sequenator. Cleaved PETMA-PTH derivatives may becollected in a fraction collector and submitted to off-linemass-spectrometric analysis utilizing a mass-spectrometer as discussedabove, or, connected on-line to the same instrument.

In certain instances, it is preferable to include a step to furtherresolve amino acids of an identical molecular weight such as leucine orisoleucine, subsequent to cleaving the derivatized amino acid from thepolypeptide. For high-sensitivity mass-spectrometric analysis of samplesinjected on-line from a separation system, the flow rate of theseparating solvent is of crucial importance. Suitable methods in thisregard include capillary electrophoresis, ion-exchange HPLC and RP-HPLC.

Capillary electrophoresis (CE) connected on-line to a mass spectrometeris a preferred method for resolving amino acid derivatives because: (1)CE has an extremely high resolving power, separations with severalmillion theoretical plates have been documented; and (2) the solventflow in CE separations is very low. The solvent flow in CE is induced bythe electroosmotic effect. As a consequence, the flow is dependent onthe pH of the solvent and additionally does not suffer from any "wall"or diffusion effects which disturb the separating power.

High-Pressure Liquid Chromatography (HPLC) or Reversed-PhaseHigh-Pressure Liquid Chromatography (RP-HPLC) (preferably with capillarycolumns) are also preferred for their ability to resolve identicalmolecular weight amino acids. Briefly, within one embodiment the cleavedderivatized terminal amino acid is eluted from the automated sequenatoras discussed above, and redissolved in 200 μl of transfer solution (seebelow). Between 10 and 100 μl per minute may be injected into anRP-HPLC, thus resolving amino acids of an identical molecular weight.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES

Unless otherwise stated, analysis of nmr spectra was performed with aBruker HC-200 Spectrometer. Analysis of mass spectra was accomplishedwith a modified AEI-MS9 mass spectrometer (Kratos, Manchester, England).Analysis of infrared spectra was accomplished with a Perkin-Elmer 1710Infrared Fourier Transform Spectrometer (Perkin-Elmer, Norwalk, Conn.).Melting point was analyzed with a Mel-Temp II (Laboratory Devices,Holliston, Mass.) melting point apparatus.

EXAMPLE 1 Preparation of PETMA-PITC (p-isothiocyanato phenethyltrimethylammonium chloride)

A. PREPARATION AND CHARACTERIZATION OF P-NITRO PHENETHYL TRIMETHYLAMMONIUM IODIDE ##STR1##

Five grams (0.025 moles) of 4-nitrophenethylamine hydrochloride(Aldrich, Milwaukee, Wis.) was dissolved in 2 ml of deionized water. Thesolution was then diluted with 15 ml of acetone, and then treated with4.6 g (0.33 mol) of K₂ CO₃ and 10 ml (0.161 mols) of iodomethane(Aldrich, Milwaukee, Wis.). The solution was refluxed for 3 days.Solvent was then removed in vacuo, and the solid that remained wasdissolved in excess hot ethanol and filtered.

The final product was recrystalized twice from ethanol to produce 7.5 g(a 91% yield). This product had a melting point of 189° C.-191° C.(compared to a literature value of 195° C.-196° C.), and produced thefollowing nmr spectra:

¹ H NMR: d(D₂ O)8.21(d, 2H, J=8 Hz) 7.5 (d, 2H, J=8 Hz) (AA¹ BB¹pattern, Ar--H) 3.60(m, 2H, ArCH₂), 3.30(m, 2H, CH₂ N), 3.18(S, 9H,N--CH₃)

B. PREPARATION OF P-AMINO PHENETHYL TRIMETHYLAMMONIUM ACETATE ##STR2##

Four grams (0.12 mols) of the product from step A, above, was dissolvedin 80 ml of 90% acetic acid. Six point four grams (0.098 mols) of zincdust was added slowly over 10 minutes, then the reaction was stirred for5 hours at room temperature. The solution was filtered and washed with70% ethanol, followed by the addition of solid Na₂ CO₃ until neutralitywas achieved. The solvents were removed in vacuo and the solid wassuspended in excess boiling methanol. The solution was hot filtered, andthe filtrate was then evaporated to dryness. Following the sameprocedure, the remaining solid was then treated with acetone instead ofmethanol.

The solid was dissolved in water, filtered, then lyophilized overnightto produce 2.3 g (81% yield) of a tan solid. This product had a meltingpoint of 200#C(d), infrared spectra of IR (KBr) 3393 (s, b), 3317(s),1633(s), 1516(s), cm⁻¹, mass spectra of M⁺ 179 (by Fast AtomBombardment-"FAB", AEI-MS9), and produced the following nmr spectra:

¹ H NMR: d(D₂ O) 7,15(d, 2H, J=8 Hz) 6.82(d, 2H, J=8 Hz) (AA ¹ BB¹pattern, Ar--H), 3.50(m, 2H, ArCH₂), 3.18(S, 9H, N--CH₃) 3.02(m, 2H, CH₂N), 1.85 (S, 3H, CH₃ COO³¹)

¹ H NMR: d(DMSO-D₆) 4.96 (S, 2H, NH₂)

C. PREPARATION OF P-ISOTHIOCYANATO PHENETHYL TRIMETHYLAMMONIUM CHLORIDE(PETMA-PITC) ##STR3##

PETMA-PITC was synthesized utilizing acetone as a solvent forthiophosgene in a procedure similar to that of Tsou (see U.S. Pat. No.3,028,397). Briefly, 100 mg (0.42 mmols) of the final product from stepB, above, was suspended in 5 ml of acetone, and treated with 0.1 ml(1.31 mmols) of thiophosgene and stirred for 2 days at room temperature.The reaction was stirred over AgX 1-8 (OH⁻) resin (Dowex, Aldrich,Milwaukee, Wis.) for 5 minutes, then quenched with methanol. Thesolution was filtered and evaporated to a brown oil. The oil waspurified by preparative thin layer chromatography utilizing a mixture ofethyl acetate, ethanol, and water (7:2:1).

The final product was a soapy white solid. The yield (30 mg, or 27.8%)was not particularly high due to the sample's retention of water (asdetected by nmr), which was not removed by vacuum. The final product hada molecular mass of M⁺ 222 (as determined by a SCIEX model API IIItriple quadrupole mass spectrometer equipped with an ion spray ionsource), and the following infrared spectra: IR(KBr) 3369(s) 2128(s)1516 (m) cm⁻. The final product produced the following nmr spectra:

¹ H NMR: d(Acetone D₆)7.60 (d, 2H, J=8), 7.35(d, 2H, J=8) (AA¹ BB¹pattern, Ar--H), 4.00 (m, 2H, Ar--CH₂), 3.56 (S, 9H, N--CH₃), 3.35 (m,CH₂ --N)

Larger preparative quantities of the final product were purified on apreparative reverse-phase HPLC column (Vydac C-18, Vydac, Hesperia,Calif.) 20×300 mm. Column buffers included 0.1% trifluoroacetic acid(TFA) in water and 0.1% TFA in a 70:30 mixture of acetonitrile andwater. The product was detected by UV absorbance of 270 nm. The peakcorresponding to the product was immediately frozen and lyophilized. Thelyophilized product, presumably the trifluoroacetate salt of thecompound, appeared as a fluffy, off-white powder and had the samephysical constants as the product purified by TLC.

EXAMPLE 2 Measurement of coupling and cleavage rates for PETMA-PITC

A 10-amino-acid decapeptide (hereinafter referred to as "ACP")containing the sequence Val Gln Ala Ala Ile Asp Tyr Ile Asp Gly (SEQ IDNO: 1) was synthesized by solid phase synthesis using an AppliedBiosystem Synthesizer (Applied Biosystems, Foster City, Calif.), andpurified by HPLC. This peptide was utilized in the following reactionsin order to determine the coupling/cleavage rate of PETMA-PITC.

A. MEASUREMENT OF THE COUPLING RATE OF PETMA-PITC AND A DECAPEPTIDE

Ten microliters of 1 mg/ml ACP, 30 μl of coupling buffer (containing 5%N-ethylmorpholine (Sigma) and 70% methanol), and 10 μl of PETMA-PITC inwater (final cone. 0.8%) were placed into a small microfuge tube, andincubated at 50° C. in a heat block.

Samples were collected after 1, 2, 15, and 30 minutes, and immediatelyinjected directly into an RP-HPLC system (Waters, Division of Millipore,Milford, Mass.) which utilized a Vydac C4 column (Vydac, Hesperia,Calif.). Column buffers included 0.1% trifluoroacetic acid (TFA) inwater, and 0.1% TFA in a 70:30 mixture of acetonitrile and water.

Elution of peptide from the column was monitored at 214 nm. The degreeof coupling was calculated based on the relative peak sizes ofunderivatized and derivatized peptides. The percentage coupling after 1,2, 15 and 30 minutes is shown below in Table 1.

                  TABLE 1                                                         ______________________________________                                        Time course of coupling reaction                                                             Coupling                                                              Time (min)                                                                            (%)                                                            ______________________________________                                               1       6                                                                     2       10                                                                    15      50                                                                    30      66                                                             ______________________________________                                    

The compound concentration (0.8%) used in this experiment was too low toachieve optimal coupling. Typically, compound concentrations in therange of 2%-5% are generally preferred. Using comparable compoundconcentrations, the coupling kinetics observed with PETMA-PITC iscomparable with the kinetics for phenylisothioscyanate (PITC), thestandard protein sequencing compound.

B. MEASUREMENT OF THE CYCLIZATION/CLEAVAGE RATE OF PETMA-PITC

A peak containing the ACP decapeptide derivatized with PETMA-PITC wasisolated and divided into four aliquots which were dried by vacuum.Individual aliquots were then exposed to 20 μl of 100% TFA, and held ata temperature of 50° C. At times of 1, 2, and 5 minutes samples werediluted with 20 μl of water, and injected into an RP-HPLC. Progress ofthe cyclization/cleavage reaction was monitored by a shift in therentention time of the peak of the starting material to a product peakwhich eluted earlier in the chromatogram. As indicated below in Table 2,cleavage was essentially complete after 2 minutes.

                  TABLE 2                                                         ______________________________________                                                       Cleavage                                                              Time (min)                                                                            (%)                                                            ______________________________________                                               1       85                                                                    2       94                                                                    5       92                                                             ______________________________________                                    

Example 3 Detection of a PETMA-PTH Amino Acid Derivatives in an IonSpray Mass-Spectrometer

Forty milligrams of PETMA-PITC (molecular weight=221.34 g/mole) wastaken up in 1.8 ml of water to provide a stock solution of 100nmoles/μl. One milligram of each amino acid (valine, alanine,isoleucine, and glycine) was placed into an eppendorf tube and dissolvedin 100 μl of a mixture of water, methanol, ethyl acetate (49:50: 1). ThepH was then adjusted to 9.2 with triethylamine, resulting in a finalamino acid concentration of 100 lmoles in 100 μl.

To each amino acid sample 10 μl of stock PETMA-PITC was added. Themixture was incubated at 50° C. for 15 minutes, and then dried byvacuum. Fifty microliters of 50% trifluoroacetic acid in water was addedto the reaction mixture, which was then incubated at 50° C. for 30minutes, and brought to dryness under vacuum. The products were thenredissolved in 200 μl of water and HPLC purified on a gradient of 0-60%B (70% acetonitrile, 30% water, 0.1% TFA) over 20 minutes on ananalytical HPLC system equipped with a Vydac C-18 column (4.6×300 mm)(Vydac, Hesperia, Calif.). The isolated purified PETMA-PTH derivativeswere checked for 25 concentration by UV spectrometry at 270 nm. Resultsare set forth below in Table 3.

                  TABLE 3                                                         ______________________________________                                        Valine        Alanine    Isoleucine                                                                              Glycine                                    ______________________________________                                        Abs.    0.40      0.41       0.27    0.55                                     Ext. Coeff.                                                                           14,000    14,000     14,000  14,000                                   c(M)    2.8 × 10.sup.-5                                                                   2.9 × 10.sup.-5                                                                    1.9 × 10.sup.-5                                                                 3.9 × 10.sup.-5                    nmoles  43        44         29      59                                       ______________________________________                                    

The purified derivatized amino acids were infused at a rate of 2 μl perminute into a Triple Quadrupole Mass-Spectrometer (API III, Sciex,Thornbill, Ontario, Canada). The mass spectrometer was equipped with anion spray ion source. The solvent for the infusion was 0.1% TFA inwater.

Results of the experiments are provided in FIGS. 1 through 8. FIGS. 1through 4 illustrate the mass spectra for isoleucine, alanine, glycine,and valine respectively. FIG. 5 illustrates the mass spectrum of valineat a 1:10 dilution (2.8 picomoles injected). FIGS. 6 through 8illustrate the mass spectra of valine at dilutions of 1:10, 1: 100, and1:1000 respectively. These figures illustrate that even at an injectedsample amount of 28 femtomoles (FIG. 8), valine can still be clearlyidentified.

Example 4 Automated Sequence Analysis Of A Peptide Utilizing PETMA-PITC

A peptide was covalently attached to a Sequelon membrane(Milligen/Biosearch, Burlington, Mass.) using the water solublecarbodiimide 1-ethyl-3-(3-dimethylamine propyl carbodiimide HCl (EDC)(Sigma), Aebersold et al., Anal. Biochem. 187:56-65 (1990). Briefly,peptide solution was applied to 1-cm circular disks of Sequelon membranein 5 μl aliquots and dried with a stream of air. Peptides were appliedeither in aqueous solution or in HPLC elution buffer, typically 0.1% TFAin H₂ O/CH₃ CN. A fresh solution of EDC (20 mg/ml in H₂ O, w/v) wasprepared immediately before use. Thirty microliters of the buffer 200 mMMES, pH 4.8 and 10 μl of EDC solution were applied to the dry disk. Thebuffer for standard coupling conditions was 200 mM MES, pH 4.5. Thecoupling reaction was allowed to proceed for 30-60 minutes at 37° C.,then the disk was extensively washed with distilled H₂ O to removeexcess EDC and any noncoupled peptide.

The following reagents are utilized in automated sequence analysis:

Washing solvent #1: 50% methanol and 50% water

Washing solvent #2: 100% acetone

Coupling base: 5% N-ethyl morpholine in methanol: H₂ O (75:25)

PETMA-PITC: 5% by weight in water

Transfer solution: 100% water or 10% acetonitrile

Cleavage solution: 100% TFA

Conversion compound: 20% trifluoroacetic acid in water

The membrane with peptide attached was first washed with 1 ml of washingsolvent #1 and 1 ml of #2, and dried with argon gas. A mixturecontaining 50 μl of coupling base and 50 μl of PETMA-PITC was deliveredto the membrane, which was then incubated for 5 minutes at 50° C.Another 100 μl of the mixture (coupling base and PETMA-PITC) was onceagain delivered to the membrane (displacing the first), and incubatedfor 5 minutes at 50° C. The membrane was then washed extensively withsolvent #1 for two minutes at a rate of 200 μl per minute. The membranewas then washed with solvent #2 for two minutes at a rate of 200 μl perminute. The membrane was then dried with argon gas. One hundredmicroliters of cleavage solution was then delivered to the membrane,which was then incubated for 3 minutes at 50° C. Displaced cleavagesolution containing the derivatized amino acid was then transferred to aconversion flask, and dried with argon gas. The solid was thenredissolved in 50 μl of conversion compound, and incubated for 15minutes at 60° C., and once again dried down with argon gas. The finalproduct was redissolved in 200 μl of transfer solution, and injectedinto an RP-HPLC.

When an HPLC separation step is utilized prior to mass spectrometry,amino acids with identical molecular weights such as leucine andisoleucine, may be distinguished. Subsequent to HPLC separation, thesample is analyzed by ion spray mass-spectrometry in a SCIEX model APIIII triple quadrupole mass spectrometer equipped with an ion spray ionsource.

Example 5 Preparation of C5 PETMA-PITC:(4-(trimethylaminopentylamidoethyl)phenyl isothiocyanate)

This example illustrates the preparation of C5 PETMA-PITC. The chemicalstructure of C5 PETMA-PITC is related to PETMA-PITC and differs only inthe length and nature of the linker which connects the isothiocyanategroup with the ionizable group. In PETMA-PITC, the linker is a phenethylgroup, while in C5 PETMA-PITC, the linker is a phenethylamidopentylgroup. The effect of the additional amidopentyl group in C5 PETMA-PITCis to render the compound more hydrophobic than PETMA-PITC. Theionizable group in both compounds is a trimethylammonium group.

The preparation of C5 PETMA-PITC may be accomplished in four stepsstarting with commercially available 6-aminocaproic acid.

A. PREPARATION OF 6-TRIMETHYLAMINOCAPROIC ACID ##STR4##

To a 500-ml round-bottom flask was added 5.0 g (49.5 mmole)6-aminocaproic acid, 5.0 g (35.9 mmole) potassium carbonate and 250 mlacetone/water solution (1:1, v/v). The stirred mixture was cooled to 0°C. in an ice bath and 10 ml (161 mmol) iodomethane was added. Thereaction was allowed to warm to room temperature, and then refluxed at65° C. for five hours at which point no starting material remained asdetermined by thin layer chromatography. The product at this stage was amixture of the quaternary trimethyl ammonium carboxylic acid and itscorresponding methyl ester. The solvent was removed from the reactionmixture in vacuo, the residue was dissolved in 100 ml 1N NaOH, andheated to 80° C for five minutes. Upon cooling to room temperature, thereaction mixture was adjusted to pH 7.5 with 12 M HCl. Removal of thesolvent by evaporation gave the crude reaction product which was usedwithout further purification. The quaternary ammonium carboxylic acidproduct was characterized by electrospray mass spectrometry. Massspectrum: m/z 174 (100, M+).

B. PREPARATION OF 4-(TRIMETHYLAMINOPENTYLAMIDOETHYL) NITROBENZENE##STR5##

To a solution of 0.50 g (2.9 mmole) quaternary ammonium carboxylic acidproduct (obtained from step A above) in 90 mldimethylformamide/dichloromethane solution (7:9, v/v) was added 2 mltriethylamine. The solution was cooled to 0° C. in an ice bath followedby the addition of 700 μl (7.3 mmole) ethylchloroformate. After stirringfor 40 minutes, 5.0 g (24.7 mmole) p-nitrophenethylamine was added andstirring was continued at 0° C. for three hours. The reaction mixturewas warmed to room temperature and the solvent was removed in vacuo toyield the crude reaction product which was used without furtherpurification. The quaternary ammonium nitrobenzene product wascharacterized by electrospray mass spectrometry. Mass spectrum: m/z 322(100, M+).

C. PREPARATION OF 4-(TRIMETHYLAMINOPENTYLAMIDOETHYL) PHENYL AMINE.##STR6##

To a stirred solution of 0.50 g (1.5 mmole) quaternary ammoniumnitrobenzene product (obtained from step B above) in 100 ml 10% aqueousacetic acid was added 1.45 g (21.7 mmole) zinc dust. After five hours,the solution was filtered to remove the solid zinc and the filtrate wasconcentrated in vacuo.

The crude product was purified by liquid chromatography on asemi-preparative, reverse-phase HPLC column (Vydac, C-18, 20×300 mm)with running buffers of 0.1% trifluoroacetic acid (TFA) in water and0.1% TFA in acetonitrile. The crude product, a purple sludge, wasdissolved in water sufficient to obtain a 1% solution. In a typicalpreparative run, 12 ml of the 1% solution was injected. The quaternaryammonium aminobenzene product was detected by absorbance at 269 nm,collected, and lyophilized to yield a white solid. The quaternaryammonium aminobenzene product thus obtained was characterized byelectrospray mass spectrometry. Mass spectrum: m/z 292 (100, M+).

D. PREPARATION or 4-(TRIMETHYLAMINOPENTYLAMIDOETHYL) PHENYLISOTHIOCYANATE (C5 PETMA-PITC). ##STR7##

To a solution of 0.10 g (0.3 mmoles) quaternary ammonium aminobenzene(obtained from step C above) in 35 ml of acetonitrile/water solution(4:3, v/v) was added 0.10 g (0.43 mmole) di-2-pyridylthionocarbonate.After stirring at room temperature for five hours, the solvent wasremoved in vacuo. The crude reaction product was characterized byelectrospray mass spectrometry. Mass spectrum: m/z 344 (100, M+).

The crude quaternary ammonium phenylisothiocyanate was further purifiedby liquid chromatography on a preparative reverse-phase HPLC column(Vydac, C-4, 20×300 mm) with running buffers of 0.1% trifluoroaceticacid (TFA) in water and 0.1% TFA in acetonitrile. The crude product wasdissolved in 30% acetonitrile in water. In a typical preparative ran, 6ml of an essentially saturated solution was injected. The quaternaryammonium phenylisothiocyanate product was detected by absorbance at 269nm, collected, and lyophilized to yield a white solid.

Example 6 Preparation and Ion Spray Mass Spectral Characterization of C5PETMA-PTH Amino Acid Derivatives

This example illustrates the preparation and mass spectralcharacterization of C5 PETMA-PTH amino acid derivatives. Four aminoacids, valine, isoleucine, alanine, and glycine, were reacted with C5PETMA-PITC to produce the corresponding phenylthiohydantoins (C5PETMA-PTH amino acid derivatives) which were subsequently purified byliquid chromatography and characterized by electrospray massspectrometry.

A stock solution of C5 PETMA-PITC was prepared by dissolving 5.0 mg C5PETMA-PITC in 100 ul of acetonitrile/water solution (1:1, v/v). Acoupling buffer, consisting of 5% N-ethylmorpholine, 70% methanol and25% water, was also prepared. Solutions of the amino acids (valine,isoleucine, alanine, and glycine) were prepared by dissolving 1.0 mgamino acid in 100 ul coupling buffer. To the amino acid solution wasadded 10 ul of the stock C5 PETMA-PITC solution followed by heating at50° C. for 15 minutes. The reaction mixture was concentrated to drynessin vacuo, 50 ul of TFA/water solution (1:1, v/v) was added, theresulting solution was heated at 50° C. for 30 minutes, and againbrought to dryness under vacuum. The crude reaction product wasdissolved in 200 ul water and purified by liquid chromatography onreverse-phase Vydac C-18 (4.6×300 mm column) with running buffersconsisting of 0.1% TFA in water (A), 0.1% TFA in acetonitrile (70:30,v/v) (B), and a gradient of 0-80% B over 30 minutes. For each of thereactions, the major product was eluted, collected, and analyzed byelectrospray mass spectrometry. The mass spectrum of each product wasidentified as the phenylthiohydantoin derived from reaction of the aminoacid with C5 PETMA-PITC, i.e., the C5 PETMA-PTH of valine, isoleucine,alanine, and glycine, respectively.

EXAMPLE 7 Preparation of PITC-311:(4-(3-pyridinylmethylaminocarboxypropyl)phenyl isothiocyanate)

This example illustrates the preparation of PITC-311,4-(3-pyridinylmethylaminocarboxypropyl)phenyl isothiocyanate. Thechemical structure of PITC-311 differs from both PETMA-PITC and C5PETMA-PITC. The ionizable group in PITC-311 is a pyridine group, and thelinker between the isothiocyanate group and the ionizable group is amethylaminocarboxypropyl group. The effect of the pyridyl group andlinker render PITC-311 hydrophobic and suitable for use in adsorptive(non-covalent) sequencing protocols, i.e., sequencing protocols in whichthe peptide to be sequenced is not covalently attached to thesolid-support prior to sequence determination.

The preparation of PITC-311 may be accomplished in three steps startingwith commercially available 4-(4-nitrophenyl)butyric acid and3-(aminomethyl)pyridine.

A. PREPARATION OF 4-(3 -PYRIDINYLMETHYLAMINOCARBOXY-PROPYL) NITROBENZENE##STR8##

To a solution of 0.50 g (2.4 mmole) 4-(4-nitrophenyl)butyric acid in 30ml dimethylformamide was added 0.5 ml of triethylamine. The solution wascooled to 0° C. in an ice bath and 0.220 ml (2.3 mmoles)ethylchloroformate was added. After 30 minutes, 0.230 ml (2.3 mmoles)3-(aminomethyl)pyridine was added and stirring was continued at 0° C.for three hours. The reaction mixture was warmed to room temperature andthe solvent was removed in vacuo. The crude product thus isolated wasused without further purification. The pyridyl nitrobenzene product wascharacterized by electrospray mass spectrometry. Mass spectrum: m/z 301(100, M+).

B. PREPARATION OF 4-(3-PYRIDINYLMETHYLAMINOCARBOXY-PROPYL) PHENYL AMINE##STR9##

To 0.69 g (2.3 mmoles) pyridyl nitrobenzene (obtained from step A above)in ml 90% acetic acid was added 2.0 g (30 mmoles) zinc dust. Afterstirring at room temperature for five hours, the mixture was filteredand the filtrate was concentrated to dryness in vacuo. The crude productthus isolated was used without further purification. The pyridylaminobenzene product was characterized by electrospray massspectrometry. Mass spectrum: m/z 270 (100, M+).

C. PREPARATION OF 4-(3-PYRIDINYLMETHYLAMINOCARBOXY-PROPYL) PHENYLISOTHIOCYANATE (PITC-3 11 ) ##STR10##

To a solution of 0.62 g (2.3 mmoles) pyridyl aminobenzene (obtained fromstep C above) in 45 ml of acetonitrile/water solution (1:1, v/v) wasadded 2.0 g (8.6 mmole) di-2-pyridylthionocarbonate. After stirring atroom temperature for thirty minutes, the solvent was removed in vacuo.The crude reaction product was characterized by electrospray massspectrometry. Mass spectrum: m/z 312 (100, M+).

The crude pyridyl phenyl isothiocyanate was further purified by liquidchromatography on a preparative reverse-phase HPLC column (Vydac, C-4,20×300 mm) with running buffers of 0.1% trifluoroacetic acid (TFA) inwater and 0.1% TFA in acetonitrile. The crude product was dissolved inacetonitrile/water solution (1:2, v/v). In a typical preparative run, 10ml of an essentially saturated solution was injected. The pyridyl phenylisothiocyanate product was detected by absorbance at 269 nm, collected,and lyophilized to yield a white solid. Purification in this mannerprovided 0.52 g of PITC-311, or an overall yield of 73% for the threesteps based on 3-(aminomethyl)pyridine.

Example 8 Manual Sequence Analysis Of A Peptide Utilizing PITC-311

In this example, the utility of PITC-311 in adsorptive amino acidsequence determination is demonstrated by sequencing the syntheticdecapeptide VQAAINYING (SEQ. ID No. 1).

Five milligrams of PITC-311 were dissolved in 100 ul of acetonitrile andwater solution (1:1, v/v). A solution containing 1 mg/ml of thesynthetic decapeptide, ACP, in water was prepared as well as thecoupling buffer solution which consisted of 5% N-ethylmorpholine, 70%methanol, and 25% water. To 10 ul of the coupling buffer solution and 5ul of the ACP solution in a microfuge tube was added 10 ul of thePITC-311 stock solution. The microfuge tube was heated at 50° C. for 15minutes followed by concentration to dryness in vacuo using a SpeedVac(Savant). The residue was treated with 20 ul 50% aqueous TFA and heatedat 50° C. for 30 min. The resulting solution was diluted 1000-fold withdistilled water and analyzed by mass spectrometry. The product wasidentified as the thiohydantoin derived from the reaction of PITC-311and valine, and confirmed that the N-terminal amino acid, valine, wascleaved from the peptide.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: YES                                                        (iv) ANTI-SENSE: NO                                                          (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ValGlnAlaAlaIleAspTyrIleAspGly                                                1510                                                                          __________________________________________________________________________

We claim:
 1. A compound comprising:(a) an isothiocyanate group; (b) anionizable group capable of detection by mass spectrometry; and (c) alinker connecting said isothiocyanate group with said ionizable group.2. The compound of claim 1 wherein said ionizable group is a stronglybasic group.
 3. The compound of claim 1 wherein said ionizable group isa strongly acidic group.
 4. The compound of claim 1 wherein saidionizable group is pyridinyl.
 5. The compound of claim 1 wherein saidlinker is hydrophobic.
 6. The compound according to claim 1 wherein saidcompound is p-isothiocyanato phenethyl trimethylammonium and counterionsalts thereof.
 7. The compound according to claim 1 wherein saidcompound is 4-(trimethylaminopentylamidoethyl) phenyl isothiocyanate andcounterion salts thereof.
 8. The compounds of claims 6 or 7 wherein saidcounterion salt is a halogen.
 9. The compounds of claims 6 or 7 whereinsaid counterion salt is a oxyanion of an acetate or trifluoroacetate.